A long range goal of this project is to predict the three-dimensional structural of proteins based on the amino acid sequences. For this understanding of major interatomic interactions which stabilize the structure is necessary. Our recent studies have shown that the cytochrome c fragment complex is stabilized by the core domain-domain interaction. A core domain consists of a hydrophobic core and the surrounding shell and folds and unfolds as a unit. Our studies have suggested that this core domain-domain interaction is mediated by the residue-residue interaction in the ordered hydrophobic core which is called the core group interaction. To know more about this interaction effect of the Leu 32 to Nva and Leu 35 to Nva substitutions and a combination of them on the stability of a horse cytochrome c three- fragment complex have been studied. The complex (1-25)H.(28-38).(39-104) contains a heme fragment of residues 1 to 25, (1-25)H and two apofragments (28-38) and (39-104). It resembles the native protein with the exception that residues 39 to 55 are flexible. The Leu 35 to Nva did not significantly decrease or only slightly decreased the apparent equilibrium constant K of (28-38) with ferric complex (1-25)H.(39-104) at 15 degree and somewhat increased that with ferrous (1-25)H.(39-104) (Kred). It did not change heat stability of the 695 nm band of ferri (1- 25)H.(28-38).(39-104), a band indicative of the Met80-S-Heme-Fe bond which is located on the left side of the heme as shown by Dickerson and Colleagues. In contrast, the Leu 32 to Nva decreased K and Kred, respectively by about 20 and 45 fold. It markedly reduced heat stability of the 695 nm band (about 0.7 kcal/mol). Perturbation of these properties by a combination of the two substitutions is similar to that by the Leu 32 to Nva alone. The rate constant for dissociation of fragment (39-104) of complex ferro- (1-25)H.(28-38).(39-104) (k), which occurs without going through ferro-(1-25)H.(39-104), was measured by the fragment exchange technique. The Leu 32 to Nva and Leu 35 to Nva, respectively increased k by 48 to 78 and 1.4 to 2.2 fold at 15 degree. Assuming that the positions of Leu 32 and Leu 35 are homologous to native cytochrome c. analysis of the data suggests that the Leu to Nva markedly perturbs a spatially long-range non-covalent interaction which exists in the ordered hydrophobic core of complex (1-25)H.(28-38).(39-104). This interaction involves gamma-methyl group of Leu 32 and the Fe-S bond and stabilizes the structure on both left and right side of the heme.